Arabidopsis α-Aurora kinase plays a role in cytokinesis through regulating MAP65-3 association with microtubules at phragmoplast midzone

The α-Aurora kinase is a crucial regulator of spindle microtubule organization during mitosis in plants. Here, we report a post-mitotic role for α-Aurora in reorganizing the phragmoplast microtubule array. In Arabidopsis thaliana, α-Aurora relocated from spindle poles to the phragmoplast midzone, where it interacted with the microtubule cross-linker MAP65-3. In a hypomorphic α-Aurora mutant, MAP65-3 was detected on spindle microtubules, followed by a diffuse association pattern across the phragmoplast midzone. Simultaneously, phragmoplast microtubules remained belatedly in a solid disk array before transitioning to a ring shape. Microtubules at the leading edge of the matured phragmoplast were often disengaged, accompanied by conspicuous retentions of MAP65-3 at the phragmoplast interior edge. Specifically, α-Aurora phosphorylated two residues towards the C-terminus of MAP65-3. Mutation of these residues to alanines resulted in an increased association of MAP65-3 with microtubules within the phragmoplast. Consequently, the expansion of the phragmoplast was notably slower compared to wild-type cells or cells expressing a phospho-mimetic variant of MAP65-3. Moreover, mimicking phosphorylation reinstated disrupted MAP65-3 behaviors in plants with compromised α-Aurora function. Overall, our findings reveal a mechanism in which α-Aurora facilitates cytokinesis progression through phosphorylation-dependent restriction of MAP65-3 associating with microtubules at the phragmoplast midzone.


Overall assessment
The key findings presented in this study, if validated by further experimentation, as described below, are novel and provide new insights into the molecular mechanisms underlying phragmoplast formation in plants.However, a conceptually similar study was published several E51@A 17> 2E B85 01= *1<<5 ;12>@1B>@E 9= -;1=B -8EA9>;>7E" D85@59= G#)C@>@1 ?8>A?8>@E;1B9>= >6 MAP65-1 was shown critical for its localization and function during cell division (Boruc et al., 2017, Plant Physiol).Although the mitotic functions of MAP65-1 and MAP65-3 are slightly different, conceptual advances made in the present study are limited.Specific points 1.The methods must be described in more detail.For example, the buffers used for protein purification and the protein concentrations in the in vitro assays have not been described.

FRAP experiments:
Is the turnover rate of MAP65-3 consistent throughout phragmoplast expansion?Otherwise, the data could be interpreted differently.The turnover rate should be examined during the early, middle, and late phases of cytokinesis.Alternatively, the turnover rate should be measured and compared at a consistent time point during phragmoplast expansion (e.g., 5 min after anaphase onset).
3. The purity of the recombinant proteins used in the in vitro phosphorylation assay was extremely low (Fig. 6B).Further protein purification methods, such as gel filtration chromatography, should be used.
4. The authors describe the cytokinesis duration as follows: "In wild-type cells, cytokinesis (from the stage of phragmoplast initiation to completion of phragmoplast disassembly) lasted approximately 20 min …a significantly longer time of 35 min during cytokinesis".This is important information for data interpretations and needs to be presented in a more quantitative manner (mean ± SD, sample size [n]).
5. The authors used three different terms to describe the phragmoplast middle plane, where cell plate assembly occurs (midzone, midline, and equatorial zone).These terms should be unified to avoid confusions unless they are intended to describe different structures.6. Arabidopsis BUB3 has been shown to regulate MAP65-3 localization (Zhang et al., 2018).Since the MAPK cascade also regulates MAP65 activity, multiple mechanisms are involved in MAP65 regulation.The manuscript should discuss how the mechanism revealed in this study is distinct from or similar to other mechanisms.7. It is not very clear which phragmoplast morphology the authors referred to as "early phragmoplast," "late phragmoplast," "ring-shaped," and "a solid disk."They should be clearly indicated in an early figure (e.g., Fig. 2).
8. Scale bars are missing in e.g., Figs.2C, 5A, 6F, 7A, S1B, and S2.In addition, molecular size information should be given next to the protein gel.9.The distinction between the cytokinetic function of MAP65-3 and other MAP65s is explained by citing REF 41-43 in the discussion section.This explanation might be moved to the Introduction, which clarifies why the authors focused on MAP65-3 rather than on any other MAP65s.10.How were the phragmoplast expansion rates quantified?Were both leading edges of a phragmoplast measured in a cell?Details on the analyses need to be clarified in the methodology section.
11. Fig. S1A, BD-AUR3: Are these panels placed in the correct order?12.Many typos and grammatical errors have been identified.Gross editing of the manuscript is necessary.
Reviewer #2 (Remarks to the Author): This work explores interaction between microtubule protein MAP65-3 and a-Aurora kinase during cytokinesis.The role of a-Aurora in cytokinesis and in phosphorylation of a close ortholog of MAP65-1 was shown before (Boruc et al., 2017).Here another member of the MAP65 gene family, MAP65-3 was shown to be a substrate for a-Aurora kinase.In addition, the authors demonstrate direct interaction between MAP65-3 and a-Aurora kinase.Despite identification of a new substrate of a-Aurora kinase is significant, it is necessary to determine the functional significance of the phosphorylation of MAP65-3 and the interaction between MAP65-3 and a-Aurora kinase by more detailed analysis of the mutant and transgenic lines.Below I provide specific suggestions.
The first major conceptual shortfall is the assumption that a-Aurora has only one substrate.This assumption has not been experimentally proven and contradicts the previous finding that a-Aurora phosphorylates MAP65-1.The authors assume that cytokinetic defects are caused by the lack of phosphorylation of two residues on MAP65-3.It is highly likely that a-Aurora phosphorylates other proteins and disruption of these events contributes to the cell division defects in aur1aur2.The Authors could and should compare phosphorylation events in control and aur1aur2 using phosphoproteomics.
Second significant conceptual weakness is the assumption that MAP65-3 only interacts with a-Aurora.It has been shown that MAP65-3 interacts with POK2 and TRAPPII (Herrmann et al., 2018;Steiner et al., 2016).Both of these proteins are essential for cytokinesis.Thus, defects in MAP65-3 localization or phosphorylation could affect interaction with these proteins.If MAP65-3 was the only substrate of a-Aurora, then the phosphomimetic version (MAP65-3DD) should be unable to rescue the map65-3 phenotype.However, Supplemental Figure 3 shows that the DD mutant rescues map65-3 root growth phenotype.It means that phosphorylation of MAP65-3 by aurora kinase may not be responsible for cytokinetic defects in aur1aur2.This outcome is more consistent with the hypothesis that phosphorylation of MAP65-3 interferes with the cell plate assembly through TRAPPII, POK2, or other components of the cell plate assembly.The authors should examine cell plate morphology in aur1aur2, AA, and DD mutants.They also should examine localization of POK1 and TRS120 in the mutant background.
Another fact that questions significance of MAP65-3 phosphorylation by a-Aurora is differences between phenotype of aur1aur2 mutant and line expressing MAP65-3AA.In aur1aur3 MAP65-3 does not localize to the phragmoplast tip (Figure 5C) whereas MAP65-3AA labels the entire phragmoplast (Figure 6C).Considering this discrepancy the authors should explore other substrates of a-Aurora kinase by comparing the phosphoproteome of Col-0 and aur1aur2.Inconsistency of experimental approaches to protein localization is a significant concern.Many localization studies are performed using chemical fixation, which is prone to artefacts.As a consequence there are several confusing results.For example, the lack of MAP65-3 localization at the phragmoplast tip in Figure 5C is a common artefact of chemical fixation.The authors should generate aur1aur2 mutants expressing MAP65-3 and tubulin markers and analysed co-localization of MAP65-3 for these experiments.It is also important to analyse co-localization of cell plate assembly markers KNOLLE and MAP65-3 on Col-0 and aur1aur2 backgrounds.Another example is Figure 6.The top row in panel F is labelled as the MAP65-3AA mutant whereas the localization pattern is identical to the MAP65-3DD mutant in Figure 6C.The bottom row in panel F is labelled MAP65-3DD, but the localization pattern is identical to the AA mutant in panel C.These comparisons should be performed in living cells during phragmoplast expansion.Expansion rate in Figure 5 was measured incorrectly.As Figure 5C shows that MAP65-3 may not always follow the phragmoplast tip, the expansion rate should be measured using a microtubule marker.
The authors should demonstrate how phosphorylation affects activity of MAP65-3.Slower turnover in the midzone in Figure 6G could be a consequence of defective cell plate assembly or association with other proteins.These possibilities should be verified.As authors already have lines expressing GFP-fusions of mutants, a simple-immunoprecipitation will provide information about interactomes of the mutants.
Analysis of interaction between MAP65-3 and a-Auraora is preliminary and requires functional characterization.The authors should examine the function of interaction between a-Aurora and MAP65-3 by complementing aur1aur2 with a-Aurora mutant that does not bind MAP65-3 and complement map65-3 with MAP65-3 mutant that does not bind Aurora A.
Association of MAP65-3 with microtubules in aur1aur2 suggests that MAP65-3 can bind parallel microtubules.However, during cytokinesis in aur1aur2 MAP65-3 binds only antiparallel microtubules.These data suggest that phosphorylation by a-Aurora changes microtubule bundling mechanisms by MAP65-3.This should be tested by determining polarity of microtubule bundles produced by the mutants and wild type MAP65-3 in vitro.
Minor weaknesses: Figure 1 has been published before and should be removed from the paper.
Response: We appreciate the critique that providing more in-depth methods is necessary for our work.We have thoroughly addressed this concern by adding additional details on "Methods" in the revised manuscript.
2. FRAP experiments: Is the turnover rate of MAP65-3 consistent throughout phragmoplast expansion?Otherwise, the data could be interpreted differently.The turnover rate should be examined during the early, middle, and late phases of cytokinesis.Alternatively, the turnover rate should be measured and compared at a consistent time point during phragmoplast expansion (e.g., 5 min after anaphase onset).
Response: We appreciate the reviewer's suggestion to examine MAP65-3 turnover at multiple time points during phragmoplast expansion and performed further experiments to confirm the consistency of MAP65-3 turnover rate.As shown in Supplemental Figure S7, the turnover rate of MAP65-3 is also reduced in aur1 aur2 plants at late stages of cytokinesis when compared with control plants.Quantitatively, in both control and aur1 aur2 cells, the half-life of fluorescence recovery after photobleaching (FRAP) at different cytokinesis stages shows little difference.Previously we chose to focus on early cytokinesis stages because at this time MAP65-3 retains a substantial disk-like signal that is straightforward to track.At late phases of cytokinesis, sometimes it can be difficult to distinguish whether regions lacking signal are caused by photobleaching or expansion of the MAP65-3 ring-edge.
3. The purity of the recombinant proteins used in the in vitro phosphorylation assay was extremely low (Fig. 6B).Further protein purification methods, such as gel filtration chromatography, should be used.
Response: To address this point, we have optimized our purification methods and achieved highly pure protein preparations, especially for AUR1 (Figure 6B).The improved reagents in kinase assays resulted in verification of our original results, supporting that MAP65-3 is a substrate of Aurora kinases.
4. The authors describe the cytokinesis duration as follows: "In wild-type cells, cytokinesis (from the stage of phragmoplast initiation to completion of phragmoplast disassembly) lasted approximately 20 min …a significantly longer time of 35 min during cytokinesis".This is important information for data interpretations and needs to be presented in a more quantitative manner (mean ± SD, sample size [n]).
Response: The duration time described here presents the cells with similar sizes shown in Figure 1A-B.In actuality, cells of different sizes undergo cytokinesis over vastly different timescales.For instance, flat cells with broader cell plate formation area always exhibit longer cytokinesis durations than narrow cells.Therefore, we prefer to quantify phragmoplast expansion rates, which are scarcely influenced by cell size, to depict cytokinesis progression.We appreciate the reviewer highlighting the need to provide quantitative details.In our revised manuscript, we have included mean data with standard deviations and sample sizes for the expansion rate analyses.
5. The authors used three different terms to describe the phragmoplast middle plane, where cell plate assembly occurs (midzone, midline, and equatorial zone).These terms should be unified to avoid confusions unless they are intended to describe different structures.
Response: We appreciate this feedback regarding inconsistent description.We have unified descriptions to "midzone" in the revised manuscript to avoid confusion.
6. Arabidopsis BUB3 has been shown to regulate MAP65-3 localization (Zhang et al., 2018).Since the MAPK cascade also regulates MAP65 activity, multiple mechanisms are involved in MAP65 regulation.The manuscript should discuss how the mechanism revealed in this study is distinct from or similar to other mechanisms.
The first major conceptual shortfall is the assumption that a-Aurora has only one substrate.This assumption has not been experimentally proven and contradicts the previous finding that a-Aurora phosphorylates MAP65-1.The authors assume that cytokinetic defects are caused by the lack of phosphorylation of two residues on MAP65-3.It is highly likely that a-Aurora phosphorylates other proteins and disruption of these events contributes to the cell division defects in aur1aur2.The Authors could and should compare phosphorylation events in control and aur1aur2 using phosphoproteomics.
Despite multiple attempts, we are unable to propagate full genomic clones for POK2.Therefore, we chose to examine Kinesin-12A, another kinesin-12 family member that also localizes to the phragmoplast midzone in a MAP65-3-dependent manner.We deliver these proteins (BUB3.1 and Kinesin-12A) thought to associate with MAP65-3, as well as TRS120, to wild-type and the aur1 aur2 mutant plants.Interestingly, we find those proteins still localize to the phragmoplast midzone in aur1 aur2 cells (Supplemental Figure S5), providing strong evidence that MAP65-3 continues to act upstream in recruiting these proteins in the aur1 aur2 mutant background.
Response: Actually, localization of MAP65-3 in aur1 aur2 cells resembles MAP65-3 AA localization in map65-3 cells.Both show wider localization at early stages of cytokinesis but mainly concentrate at phragmoplast midzone at late stages of cytokinesis.In Figure 6C, we aim to show that MAP65-3 AA is retained in the interior region of the ring-shaped phragmoplast, though it appears to also label the leading tip.This is because the cell imaged is not at a very late stage of cytokinesis, so the phragmoplast has not yet become a fully rounded ring.Our new live-cell imaging data confirms that MAP65-3 AA does not label the leading phragmoplast tip at late stages of cytokinesis (Figure 6H).
Inconsistency of experimental approaches to protein localization is a significant concern.Many localization studies are performed using chemical fixation, which is prone to artefacts.As a consequence there are several confusing results.For example, the lack of MAP65-3 localization at the phragmoplast tip in Figure 5C is a common artefact of chemical fixation.The authors should generate aur1aur2 mutants expressing MAP65-3 and tubulin markers and analysed co-localization of MAP65-3 for these experiments.It is also important to analyse co-localization of cell plate assembly markers KNOLLE and MAP65-3 on Col-0 and aur1aur2 backgrounds.Another example is Figure 6.The top row in panel F is labelled as the MAP65-3AA mutant whereas the localization pattern is identical to the MAP65-3DD mutant in Figure 6C.The bottom row in panel F is labelled MAP65-3DD, but the localization pattern is identical to the AA mutant in panel C.These comparisons should be performed in living cells during phragmoplast expansion.
Response: We agree additional experiments using live-cell imaging are needed to conclusively determine MAP65-3 localization.To address this point, we deliver a pTUB6:mCherry-TUB6 marker to our lines and perform live-cell confocal imaging of MAP65-3-GFP and mCherry-tubulin simultaneously.Live-cell confocal imaging of these lines reveals that MAP65-3 in aur1 aur2 background or MAP65-3 AA in map65-3 background retains in the interior region of the ring-shaped phragmoplast but less decorates its leading edge (Figure 4E, Figure 6H).These new live-cell imaging results are consistent with our original immunofluorescence findings.We have also analyzed co-localization of MAP65-3 and the cell plate marker FM4-64 via live-cell imaging (Figure 5-7).Again, MAP65-3 AA signal predominantly labels the interior region of the mature cell plate (Figure 6J).
The original FRAP experiments for MAP65-3 AA shown in Figure 6F is conducted at a late phragmoplast stage, while MAP65-3 DD is imaged at an early stage, leading to an impression that MAP65-3 AA and MAP65-3 DD have similar localization patterns.Although the turnover rate of MAP65-3 is consistent between the early and late phases of cytokinesis (Supplemental Figure S4), we have updated the MAP65-3 AA FRAP figures to an earlier cytokinetic phase to avoid any confusion (Figure 6F).Based on clarification of our original results and additional live-cell imaging experiments, we believe the seemingly inconsistencies are now fully addressed.
Expansion rate in Figure 5 was measured incorrectly.As Figure 5C shows that MAP65-3 may not always follow the phragmoplast tip, the expansion rate should be measured using a microtubule marker.
Response: We appreciate the reviewer raising this astute observation and suggestion.To address this point, we have generated MAP65-3 AA/DD lines co-expressing mCherry-tubulin to mark microtubules in the same cells (Figure 6H).In these lines, we measured the phragmoplast expansion rate using the MTs signal.These new measurements confirm our original findings that the expansion rate of the phragmoplast is significantly decreased in the MAP65-3 AA line when compared to the MAP65-3 and MAP65-3 DD lines (Figure 6I).

Minor weaknesses:
Figure 1 has been published before and should be removed from the paper.
Response: We have moved Figure 1 to Supplemental Materials.
The authors have addressed all the technical concerns raised by this reviewer; I believe that the manuscript is publishable in its current form.Meanwhile, I hold my initial view that the conceptual advances made in this study are marginal and the study falls short of the level of Nature Communications.
Reviewer #2 (Remarks to the Author): This is a revised manuscript that I reviewed several months ago.In response to my comments the authors performed additional experiments and modified the text.I appreciate the efforts in providing additional experimental data and explanations.
Although this version includes a sizable dataset, papers published in Nature Communications are supposed to address mechanisms of biological processes by providing a substantial body of experimental data.In my opinion, the existing data in inconclusive mostly because my major criticism was not addressed directly though these experimental are relatively fast and simple.Additional experimental evidence is necessary to answer the question about the role of phosphorylation of MAP65-3 by Aurora.
It has been shown that MAP65-3 plays two main roles: bundling anti-parallel microtubules and targeting other proteins to the midzone.Before the manuscript is published in Nature Communications the authors should perform experiments that answer which of the functions or both are affected by the phosphorylation.
The first major concern about the dataset is that MAP65-3 still localizes to the midzone in the aur1aur2 as in the Col-0.The only phenotype is slower turnover of MAP65-3 in the midzone.However, this phenotype is unlikely to be a consequence of interaction between MAP65-3 and microtubules because as shown in Figures 3 and 4, MAP65-3 remains in the midzone even after depolymerization of microtubules.This fact means that phosphorylation controls retention of MAP65-3 in the midzone through interaction with other proteins.The author analyzed localization of several known MAP65-3 interactors and midzone makers such as BUB3.1,Kin-12A, TRS120, and Knolle.All these proteins showed normal localization.However, there are many midzone proteins that were not tested.The authors should compare interactomes of MAP65-3, MAP65-3DD, and MAP65-3AA.This analysis will reveal which protein could be responsible for keeping MAP65-3 in the midzone after depolymerization of microtubules.
The third critical concern is the claim that phosphorylation by Aurora restricts activity of MAP65-3.However, the phragmoplast expands normally.Further, MAP65-3DD can rescue map65-3 mutant phenotype but can not rescue aur1aur2 phenotype.The data provided in the manuscript strongly supports hypothesis that MAP65-3DD mutant functions normally and phosphorylation does not affect its activity.The authors should perform direct functional assays by test the impact of MAP65-3DD on microtubule dynamics and bundling in vitro and in vivo.These data will determine whether phosphorylation restricts or increases activity of MAP65-3.
We sincerely appreciate the reviewer providing valuable feedback and insightful critiques on our initial revised manuscript.In response to these comments, we have conducted additional experiments comparing the MAP65-3 interactome and phosphoproteome between WT control and aur1 aur2 plants.We have also made the suggested revisions to the manuscript text and figures based on the reviewer's recommendations, as detailed in our point-by-point responses below.
Reviewer #2 (Remarks to the Author): This is a revised manuscript that I reviewed several months ago.In response to my comments the authors performed additional experiments and modified the text.I appreciate the efforts in providing additional experimental data and explanations.
Although this version includes a sizable dataset, papers published in Nature Communications are supposed to address mechanisms of biological processes by providing a substantial body of experimental data.In my opinion, the existing data in inconclusive mostly because my major criticism was not addressed directly though these experimental are relatively fast and simple.Additional experimental evidence is necessary to answer the question about the role of phosphorylation of MAP65-3 by Aurora.
It has been shown that MAP65-3 plays two main roles: bundling anti-parallel microtubules and targeting other proteins to the midzone.Before the manuscript is published in Nature Communications the authors should perform experiments that answer which of the functions or both are affected by the phosphorylation.
The first major concern about the dataset is that MAP65-3 still localizes to the midzone in the aur1aur2 as in the Col-0.The only phenotype is slower turnover of MAP65-3 in the midzone.However, this phenotype is unlikely to be a consequence of interaction between MAP65-3 and microtubules because as shown in Figures 3 and 4, MAP65-3 remains in the midzone even after depolymerization of microtubules.This fact means that phosphorylation controls retention of MAP65-3 in the midzone through interaction with other proteins.The author analyzed localization of several known MAP65-3 interactors and midzone makers such as BUB3.1,Kin-12A, TRS120, and Knolle.All these proteins showed normal localization.However, there are many midzone proteins that were not tested.The authors should compare interactomes of MAP65-3, MAP65-3DD, and MAP65-3AA.This analysis will reveal which protein could be responsible for keeping MAP65-3 in the midzone after depolymerization of microtubules.
Response: We want to clarify that the retention of MAP65-3 is always coincident with residual microtubules at the phragmoplast midzone in aur1 aur2 cells, based on our observations.At intermediate stages of cytokinesis, these incompletely depolymerized microtubules are difficult to discern due to the overwhelming fluorescence signal from the high density of microtubules in neighboring areas.However, low amounts of undepolymerized microtubules are present in the interior region when the phragmoplast first transitions to a ring shape.We apologize for the confusion due to not capturing later cytokinesis stages showing full microtubule disassembly in our main figures.As shown in Movie S10, once phragmoplast microtubules are completely depolymerized at late cytokinesis (over 30 min of cytokinesis), almost all MAP65-3 signal no longer remains at the interior phragmoplast midzone in aur1 aur2 cells (see below figure).The delayed MAP65-3 turnover we observe in aur1 aur2 cells is coincident with residual microtubules that fail to fully disassemble.We propose the slower detachment of MAP65-3 from these The revised manuscript includes three novel datasets: (i) phosphorylated residues of MAP65-3 in aur1aur2 background and in Col-0 control; (ii), interactome of MAP65-3 in aur1aur2 and in Col-0 control; and (iii) turnover rate of MAP65-3, MAP65-3AA and MAP65-3DD in the interphase cells.I much appreciate the efforts to produce these datasets.The authors use this evidence to support the hypothesis that Aurora kinase regulates cytokinesis by reducing affinity between MAP65-3 and microtubules in the midzone.
My major concern is that numerous experimental data contradict this conclusion.
1.The phragmoplast looks normal in aur1aur2 in Figure 3D, Figure 4C, E, Figure 5B, and Supplemental Figure 5A-C.Somewhat wider phragmoplast midzone in Figure 1B could be a natural variability of the phragmoplast morphology.In fact the midzone of the phragmoplast in Col-0 in Figure 1A seems wider in the time frames 4:00 and 13:40.The phragmoplast branching phenotype is only shown in Figure 5.The lower frequency of the phenotype questions whether Aurora kinase contributes to regulation of phragmoplast morphology.
Response: We wish to clarify that we did not aim to propose a severely disrupted or disorganized phragmoplast structure in aur1 aur2 plants.Rather, our results seek to demonstrate that the process of phragmoplast expansion appears obstructed or hindered in the mutant compared to WT plants.Upon those mentioned figures, we believe the key conclusions supported by our results are: (1) Phragmoplast expansion is significantly slower in aur1 aur2 compared to WT based on live-cell imaging analysis in Figure 1; (2) MAP65-3 displays altered dynamics at phragmoplast microtubule arrays in aur1 aur2 cells, with preferential accumulation at trailing edges and persistent association at dismantling interiors seen in Figures 3-5.The wider midzone and disorganized leading edges are often observed when phragmoplast expands to a matured ring-shape stage, rather than as a general defect.We have already pointed these observations when mentioned Figure 1 in the Results part.Furthermore, as shown in Figure 1 and Supplementary Movie 1, quantification of the phragmoplast midzone width over time demonstrate that, unlike in aur1 aur2 cells, the gap width remains largely unchanged in WT cells as the phragmoplast expanding centrifugally.
In Figure 5, our objective is to demonstrate the consistent enrichment and persistence of MAP65-3 signal specifically at the branch zones of a mature cell plate within aur1 aur2 plants.We also asserted that only a small subset of aur1 aur2 (*006 *;-.'.7*) '5&2(-*) (*00 40&7*6> &2) ).6(866*) 7-* .27*540&<'*7:**2 =@"8535& and membrane remodeling in our previous revision.It's important to note that the main aim of Figure 5 is not to over-interpret this less frequent branching phenotype, but rather to showcase how the turnover dynamics of MAP65-3 are altered during cytokinesis, resulting in its preferential accumulation at the interiors of mature cell plates.

Figure
Response: Regarding Figure 4C and E, the MAP65-3 localization patterns are not in fact similar between control and aur1 aur2 plants: " In control cells during late cytokinesis when the phragmoplast is dismantling at the center, MAP65-3 cleanly removes from the interior region where shows invisible microtubule fluorescence." By contrast, in aur1 aur2 cells MAP65-3 persistently associates within the dismantling interior zone, indicating it fails to timely disengage from the remnant phragmoplast structure." Additionally, at earlier timepoints MAP65-3 decorates microtubule edges more uniformly across the phragmoplast midzone in control cells, whereas in aur1 aur2 cells it abnormally accumulates in trailing edges of the ring-shaped phragmoplast.
The distinct variances in MAP65-3 distributions between the two lines corroborate our model, suggesting that altered phosphorylation affects its timely turnover during phragmoplast expansion.
3. Figure 4D shows localization of MAP65-3 at the central part of the phragmoplast lacking microtubules.Figure 4E 12' time frame also shows MAP65-3 in the region devoid of microtubules.In the response to my comment, the authors claim that this region contains remnant microtubules, these microtubules are invisible on the images provided though.If these regions contain microtubules, then it is important to provide relevant supporting information.Unless this data is provided, there is still a possibility that the phosphorylation controls interaction of MAP65-3 with other proteins or phosphorylation of other proteins determines localization of MAP65-3.
Response: We apologize for any confusion caused by implying MAP65-3 is permanently localized in microtubule-free regions.Upon re-examining Movie S10 over a longer timeframe of phragmoplast expansion (over 30 minutes), the MAP65-3 signal does eventually disengagement from the central region in aur1 aur2 cells.However, this process is delayed compared to control cells, demonstrating slower turnover rates.The intent of Figure 4 was to illustrate these kinetic delays.Additionally, contrary to the reviewer's observation, residual microtubules at the phragmoplast center are indeed visible at the 12' timepoint in Figure 4E (arrows), indicating a stalled rather than fully absent maturation of the ring-shaped structure in aur1 aur2 cells.The delayed maturation of the phragmoplast in aur1 aur2 cells is likely due to the slowed movement of MAP65-3 out of the central region toward the leading edges.
It should also be noted that the interdigitated microtubules at the phragmoplast midzone where MAP65-3 localized appears as an invisible dark line under both live-cell fluorescence imaging and immunofluorescence microscopy.It is known that the phragmoplast midzone region contains a significantly smaller number of interdigitated microtubules compared to the periphery of the phragmoplast.This sparse density of residual midzone microtubules makes them relatively faint and difficult to discern visually, thereby imparting a dark line appearance.We suppose there are likely still residual interdigitated microtubules present that fail to dismantle timely caused by impaired MAP65-3 turnover kinetics in aur1 aur2 cells, even as the bulk of microtubules depolymerize radially at the distal phragmoplast zone.
Our earlier proteomics experiments did not find any significant changes in MAP65-3 interacting proteins regulated by Aurora phosphorylation, but we acknowledge it is challenging to thoroughly screen for proteins specifically expressed during cell division using current techniques.Because such proteins may only be present in low amounts limited to mitotic/cytokinetic stages, rather than throughout the cell cycle, they can be difficult to detect.A thorough investigation of additional phosphorylation targets and binding partners that may contribute to MAP65-3 localization is certainly warranted but beyond the intended scope of the present study.As the reviewer insightfully points out, we cannot definitively rule out the possibility that MAP65-3 phosphorylation may also influence its interactions with other binding partners besides microtubules.In our revised discussion section, we have pointed out that MAP65-3 phosphorylation could also modify its associations with other proteins, which may contribute to the observed phenotypes and deserves further study.
4. Localization of MAP65-3 interacting proteins is affected in aur1aur2.Both BUB3.1 and Kin12-A localize in the region devoid of microtubules (Supplemental Figure S5).This outcome provides a very strong support that MAP65-3 is retained in the midzone after microtubule depolymerization due to interaction with BUB3.1, Kin12A, or both, but not because of stronger affinity with microtubules.
5. If constitutively active MAP65-3 was responsible for the cytokinetic defects in aur1aur2, then the ectopic expression of MAP65-3AA would cause similar defects.However, phragmoplast morphology was not affected in cells expressing MAP65-3AA as shown in Figure 6H.
#3:*9*5> & (036*5 033/ &7 7-* 7.1*@0&46* )&7& .2 Figure 6H does reveal a subtle phenotype of prolonged phragmoplast microtubule arrays in the 18:20 time frames of MAP65-3 AA -expressing cells compared to earlier time points.This phenocopies what is observed in aur1 aur2 plants.More importantly, expressing MAP65-3 AA in map65-3 cells causes similar delayed phragmoplast expansion effects as seen in aur1 aur2 cells (Figure 6I).Additionally, the microtubule distribution patterns and turnover dynamics of MAP65-3 AA once again mimic what is observed in the aur1 aur2 mutant background.(Figure 6C, F).Reviewer #2 (Remarks to the Author): This is the fourth version of the manuscript by Deng et al.The textual changes to this version have increased the clarity and my concerns were addressed.Overall, the manuscript was significantly improved since the first submission.